The objective of this investigation is the development and application of biophysical methods to separate and isolate homogeneous tumor cell populations from solid tumors in order to characterize in situ their response to radiation and other deleterious agents. Using density gradient centrifugation, L-P59 sarcoma cells were effectively separated into four subpopulations following in situ irradiation. The Do's of these populations ranged from 240 to 330 rads with the most dense population being the most radiation resistant. Experiments were conducted to determine the effects, if any, of 5-thio-D-glucose on hypoxic fibrosarcoma cells in situ. No cytotoxic or sensitizing effect was observed in either the unseparated or density separated tumor population. The method of centrifugal elutriation was also characterized and applied to separate and synchronized tumor cells. The cloning efficiencies of these various synchronized populations were tested using a lung colony assay. No effect of either cell size or position in the cell cycle on colony forming efficiency was observed. We will continue to separate cells from tumors by either density and/or centrifugal elutriation following in situ treatment, and will characterize them with respect to: a) response to radiation sensitizing electron-affinic drugs, b) ability to accumulate and repair sublethal and/or potentially lethal drugs; and c) age response to radiation and other deleterious agents.